New parameters for the in vitro development of cell lines from fish species

The establishment of cell lines from fish species is becoming important because of the increase in interest of these cells for viral analysis, environmental monitoring and cytogenetic studies. However, there is some discussion about the best conditions for maintaining these cells. We describe here a protocol for primary cell lines which we have found to be reliable. Fin biopsies from two species, Geophagus proximus and Astyanax bimaculatus , were isolated and cultured. We used three culture media (Leibovitz-L15, Dulbecco s Modified Eagle Medium-DMEM and 199) with or without the addition of AmnioMax and a standard temperature of 29ºC. The results showed that 199 medium was less efficient for both species. However, the cells of G. proximus had better proliferation in DMEM and L-15 media, while A. bimaculatus cells fared better in DMEM medium. The high concentration of L-glutamine and branched-chain amino acids (BCAAs) in the DMEM and L15 media was probably important for better adaptation of these cells. Furthermore, the addition of AmnioMax, a supplement rich in L-glutamine, increased cell proliferation in both species. Thus, the protocol initially established was tested in other tissues (fin, gills and kidney) of other fish species from the Amazon region. The cells were maintained in L-15 medium supplemented with 20% FBS (Fetal Bovine Serum) and 5% AmnioMax. It was observed that the cells were successfully subcultured and had a good proliferation, also the morphological characteristics were preserved. Thus, the methodology described in this research represents an innovative tool for the establishing of fish cell.